Factors affect the structure of plexin-B1 and plexin-C1

Buck Lab

Abstract:Plexins are semaphorin receptors that play essential roles in many biological processes. Plexin signaling relies on a semaphorin-induced dimerization mechanism and is regulated by small GTPases of the Rho family. In addition, the binding of small Rho GTPases with the Rho GTPase Binding Domain (RBD) of plexin is necessary to the function of plexin. There are a couple of cysteine in RBDs, and cysteine can form intermolecular disulfide bonds. It is believed that disulfide bonds can modulate the structure and the function of RBDs. Understanding what factors can affect the formation of disulfide bonds can help us decipher the structure and the function of plexin. In the research, plexin-B1 and plexin-C1 proteins, different pH values buffers, an oxidizing reagent (_H2O2) and two reducing agents (β-ME, or DTT), are used to study the structure of plexin. SDS-PAGE is used to determine the molecular weight of the protein. ¹H- ¹⁵N HSQC is utilized to investigate the chemical shift perturbation, helping understand the structure of plexin. The results show that the protein prefers to form a dimer, trimer, or oligomer in some specific conditions. When the dimer forms, protein-protein interaction happens between the dimer interfaces. The effect leads to some missing peaks in NMR spectra. This analysis of interaction provides an approach to help understand the structure and the function of plexin.

Pharmacological activation of unfolded protein response promotes proteostasis of  epilepsy-associated GABAA receptors 

Mu Lab

Protein homeostasis between GABA_A receptor folding, trafficking and degradation is essential to  ensure normal physiological functions. Mutations in GABA_A receptors lead to numerous neurological disorders, including genetic epilepsy, and many patients suffering from which are  resistant to current drug treatments. Therefore, developing novel therapeutic strategies to target  defective GABA_A receptors is critical to effectively treat genetic epilepsy. In this study, we used  HEK293T cells that exogenously express disease-associated variants in the β2 subunit of  GABA_A receptor (i.e., I246T) with wild type α1 and γ2 subunits. Cell surface biotinylation assay  demonstrated that these β2 variants result in significantly reduced surface expression of the β2  protein compared to the wild type, mainly by decreasing folding and surface trafficking of the  mutant receptors. Automated patch clamping experiments further showed that these β2 mutants  have significantly diminished current compared to the WT receptor. Additionally, we searched  for small molecules that rescue the functional surface expression of the pathogenic receptors.  We found that GABA_A receptors-specific pharmacological chaperones restored the surface  expression of these β2 mutants. Western blot analysis further showed that pharmacologically  activating the unfolded protein response enhanced the total and surface protein levels of β2  variants. Overall, our results demonstrated that these compounds, while having distinct  mechanisms of action, hold great therapeutic potentials to treat genetic epilepsy by targeting the  disease-associated GABA_A receptor variants. 

Investigating the effect of GABRA1 frameshift mutations on GABAAR function

Mu Lab

Abstract:Frameshift mutations in the GABRA1 gene of the gamma-aminobutyric acid type A receptor  (GABA_AR) have been commonly linked to epilepsy disorders, particularly childhood absence  epilepsy and epileptic encephalopathy. However, due to its rarity and de novo nature, there is little  literature found about the disease mechanisms that underlie these mutations. Because these  mutations have substantial structural effects on the GABA_AR α1 subunit through an introduction  of a premature stop codon for translation, they have been mostly regarded as pathogenic despite  their heterozygosity. We hypothesize that the frameshift mutant α1 subunit presents as a severely  misfolded protein with trafficking defects causing accumulation in the endoplasmic reticulum  (ER) and defective GABA_AR function. Focusing on four particular frameshift mutations: K401,  S326, V290, and F272, protein overexpression and trafficking of the GABAAR α1 subunit were examined in a transiently transfected HEK293T cell model. Total and surface protein expression  of the mutants differed from the wild-type (WT) indicating defects in trafficking. Additionally,  automated patch clamp recordings revealed functionally defective GABA_AR with frameshift  mutations in GABRA1. Investigation of these mutations’ effect on assembly, trafficking, and  function will provide critical insights necessary to understanding the mechanisms of the epilepsy linked disorders as well as reveal potential therapeutic targets and pathways that could advance  current treatment research. 

PR-B Proteolytic Cleavage as a Mechanism for Functional Progesterone Withdrawal

Mesiano Lab

Progesterone (P4) is the most important hormone for the maintenance of pregnancy, and its  withdrawal, whether functional or systemic, initiates parturition. Targeting factors that lead to  inflammatory events within Progesterone Receptor (PR) pathways may help keep a fetus at risk  for preterm birth (PTB) in the uterus longer, increasing chances of survival and decreasing  potential negative consequences. However, gaps in our understanding of these highly complex  progesterone signaling pathways remain.  Canonically, once liganded, the full length PR (PR-B), is known to have anti-inflammatory  actions by interfering with AP-1’s transcriptional activity. This is a major pro-gestational drive  during the quiescent period of pregnancy. However, the anti-inflammatory activity associated  with PR-B decreases drastically as pregnancy nears its culmination—the mechanism of which is  currently unknown.   We postulate that when an “Inflammatory Threshold” is met during pregnancy, PR-B’s activity  may decline due to proteolytic cleavage. This cleavage likely occurs near the 164th residue of  PR-B, directly distal to the B-Upstream Segment (BUS) that is unique to PR-B. We hypothesize  that this proteolytic cleavage, for all intents and purposes, deactivates PR-B’s anti-inflammatory  action, allowing for uninhibited pro-inflammatory signaling and pro-labor mechanisms.   This research addresses a major physiological knowledge-gap, i.e.: the mechanism by which  pregnancy is both maintained through quiescence and concluded through parturition. The goal of  this study is to determine the mechanism of PR-B cleavage and its association with the onset of  labor. This will allow us to understand the function of the progesterone receptors in the human  myometrium, and provide specific, targetable mechanisms for potential preterm birth therapies. Ultimately, this research will contribute to the development of effective PR-based therapies to  prevent PTB.  These aims will be achieved using several innovative model systems including genetically  modified human myometrial hTERT cell lines, frozen/fixed and explant cultures of gravid  human myometrium, mouse models of inflammation-induced PTB and physiological pregnancy  phenotypes, and optimized assays using validated reagents to measure 1) relative abundance of  specific mRNAs; 2) abundance of specific proteins; 3) physical interaction pathways between  PRs and AP-1 subunits; 4) specific sub-cellular localizations of PR-B; and 5) development of  model systems in human myometrial cell lines and transgenic mice, within which PRs are  mutated to observe functionality. 

Structural Studies of Amyloid Fibrils in Rapidly Progressive Alzheimer's Disease

Surewicz Lab

Abstract:Amyloid fibrils are misfolded protein filaments often associated with neurodegenerative diseases. For instance, the propagation of neurofibrillary tangles containing tau filaments within the brain contributes to the progression of Alzheimer’s Disease (AD) and is an important biomarker for the disease. To further understand the molecular basis of this propagation, we purified tau filaments from the brains of rapidly progressive AD (rpAD) patients and determined their structures using the method of cryo-EM. From the cryo-EM structures, we discovered unique structural features that can distinguish them from those of normal, slowly progressing AD patients. Such filaments will likely behave differently when interacting with many commonly used PET ligands and drugs, making it essential to develop new diagnostic and therapeutic approaches for rpAD patients. Another important aspect of our study is to understand how these self-propagating filaments are transported within the brain. Thus, we attempted to establish a system to monitor the transportation of tau filaments within neuronal axons using cryo-ET. This approach will be complementary to the high-resolution cryo-EM studies, providing additional information about the molecular mechanism of the progression of AD.

GEphA2-ephrinA interaction plays a multifaceted regulatory role in prostate cancer (PCa) development and malignant progression toward late stage PCa

Bing-Cheng Wang Lab

Abstract:Prostate cancer (PCa) is the most common cancer in the US men. While usually indolent or benign, a small fraction (~5%) rapidly progress to malignant diseases. PCa is initially responsive to androgen deprivation therapy (ADT) or castration. However, aggressive forms of the disease inevitably become resistant to the therapy, leading progressively to metastatic castration resistant PCa (mCRPC), a fraction of which further progress to neuroendocrine prostate cancer (NEPC) and double negative PCa (DNPC). A major goal of PCa research is to broadly identify molecular and cellular mechanisms aiding nearly inevitable progression to identify vulnerabilities that could be targeted. Multiple receptor tyrosine kinases (RTKs) have been implicated in PCa. A significant body of literature points to an important role of EphA2, a member of the Eph subfamily of RTKs, in PCa. Notably as first reported by Chinnaiyan lab, EphA2 RTK is overexpressed in metastatic CRPC, but not early localized PCa tumors. In tumors where EphA2 is overexpressed, there is loss of the cognate ligand EphrinA1. In fact, Colm Morrissey was the first to discover that EphrinA1 is one of the top three genes whose expression is lost in metastatic PCa, particularly in bone metastases. The Wang lab, a leading group in studying Eph/Ephrin system in cancer biology, discovered that EphA2 has dual opposed roles during tumor development and progression, i.e., a ligand dependent tumor suppressor in the early stage of tumorigenesis and a ligand independent oncogenic protein in the late stage tumor progression in several cancer types. Our preliminary data indicate that in PCa, EphA2-EphrinA signaling indeed has a tumor suppressive role in early-stage PCa, and tumor promoting role in metastatic CRPC. Excitingly, our studies show potential for regulation of EphA2 expression by androgen receptor (AR) function. Our current hypothesis is that EphA2-ephrinA interaction plays a multifaceted regulatory role in prostate cancer (PCa) development and malignant progression toward late stage PCa. The outstanding questions are addressed by examining EphA2 expression across human PCa samples, and modeling PCa progression using in vitro and in vivo systems.

ATAD3A is indispensable for retinal cell health and vision

Qi Lab

Abstract: Accumulating evidence suggests that many neurological disorders, especially those which affect mitochondria, are correlated with vision loss. The retina contains a network of post-mitotic neurons to process vision, and any damage or disruption to this complex cellular network can result in partial or total blindness. In our study, we examined the role of ATAD3A: a mitochondrial membrane protein which has been identified to have preliminary association with inherited retinal and optic nerve disorders. Familial studies have identified many different mutations in ATAD3A which alter or inhibit its functions. In order to understand the consequences of ATAD3A deficiency, we employed single-neuron labeling with inducible cre-mediated knockout (SLICK) in a transgenic mouse model. With this method, we are able to specifically knockout ATAD3A in projection neurons which extend to the retina. Our results suggest an indispensable role of ATAD3A retinal cell health and maintenance. By characterizing the nature of these vision-related disorders we will become better equipped to treat them and improve the quality of life of those afflicted by them.

Perturbation of Azurophilic Granules via Lysosomotropic Agents Permits Inflammasome-independent IL-1β Processing and Release

Dubyak Lab

Abstract: IL-1β is an inflammatory cytokine mainly secreted by myeloid cells in response to infection or sterile tissue damage. Non-canonical secretion of IL-1β from macrophages downstream of activated NLRP3/caspase-1 inflammasomes is the best-characterized model; this is mediated by caspase-1 cleavage of GSDMD allowing N-GSDMD to form pores in the plasma membrane that act as conduits for IL-1β release and inducers of pyroptosis as a lytic cell death. The NLRP3 initiator acts as a sensor of perturbed cellular homeostasis including decreased cytosolic [K+]. In macrophages, this K+ efflux mediated NLRP3 activation is also triggered by agents that disrupt lysosomal integrity. While neutrophils also assemble competent NLRP3 inflammasomes and release bioactive IL 1β via GSDMD-dependent mechanisms, they resist the formation of plasma membrane N-GSDMD pores and progression to pyroptosis. We tested whether lysosome disrupting stimuli in neutrophils would phenocopy macrophage mechanisms to secrete IL-1β in an NLRP3 inflammasome and GSDMD-dependent manner. Surprisingly, our data indicate that prolonged stimulation with lysosomotropic stimuli induce neutrophils to release mature IL-1β and undergo lytic cell death independently of the NLRP3 inflammasome. However, we observed that early time points following stimulation induces IL-1β release in an NLRP3 inflammasome dependent manner. Using a chemical tool (Leu-Leu-OMe) that is capable of rapidly disrupting lysosomal membranes, we observed an NLRP3 inflammasome-independent IL-1β release, mimicking prolonged stimulation. Considering the granular phenotype of neutrophils, we hypothesize that these lysosomotropic stimuli are being sequestered into the lysosome-like azurophilic granules and causing release of multiple serine proteases that are known to directly cleave IL-1β and GSDMD. To test this hypothesis, pre-treatment with serine protease inhibitors were able to suppress IL-1β release from stimulated neutrophils. Therefore, we propose that disruption of azurophilic granules coordinately disrupts canonical NLRP3 inflammasome assembly and directly cleaves proIL-1β and GSDMD as part of a novel neutrophil-specific signaling mechanism for lysosomal disruption-induced processing and export of IL-1β.

Adapting the endoplasmic reticulum proteostasis rescues epilepsy-associated NMDA receptor variants

Mu Lab

Abstract: The GRIN genes encoding N-methyl-D-aspartate receptor (NMDAR) subunits are remarkably intolerant to variation. Many pathogenic NMDAR variants result in their protein misfolding, inefficient assembly, reduced surface expression, and impaired function on neuronal membrane, causing neurological disorders including epilepsy and intellectual disability. Here, we investigated the proteostasis maintenance of NMDARs containing epilepsy-associated variations in the GluN2A subunit, including M705V and A727T. In the transfected HEK293T cells, we showed that the two variants were targeted to the proteasome for degradation and had reduced functional surface expression. We demonstrated that the application of BIX, a known small molecule activator of an HSP70 family chaperone BiP (binding immunoglobulin protein) in the endoplasmic reticulum (ER), dose-dependently enhanced the functional surface expression of the M705V and A727T variants in HEK293T cells. Moreover, BIX (10 μM) increased the surface protein levels of the M705V variant in human iPSC-derived neurons. We revealed that BIX promoted folding, inhibited degradation, and enhanced anterograde trafficking of the M705V variant by modest activation of the IRE1 pathway of the unfolded protein response. Our results suggest that adapting the ER proteostasis network restores the folding, trafficking, and function of pathogenic NMDAR variants, representing a potential treatment for neurological disorders resulting from NMDAR dysfunction.

Glycolytic Dysregulation in Amyotrophic Lateral Sclerosis

Qi Lab

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by upper and lower motor neuron death. Although the mechanism resulting in motor neuron death is unknown, alterations of the metabolic landscape has been a large area of focus. Reports of malfunctioning mitochondria in motor neuron diseases (MNDs) promotes researchers to begin looking at metabolic pathways adjacent to mitochondria. Here, we utilize in vitro and in vivo techniques to determine the changes of glycolysis in TDP43 related ALS. We found that TDP43 ALS results in reduced glycolytic output in TDP43NLS, a mutant promoting cytoplasmic mislocalization of the nuclear binding protein. Further, silencing of glycolytic enzymes results in increased cell death in both TDP43-WT and TDP43-NLS. Utilizing another mutant present in ALS patients, TDP43-A315T, we found a decrease of total protein levels of the first rate limiting enzyme within the glycolysis pathway hexokinase 1 (HK1). Subsequently, we also found a reduction of HK1 in TDP43-A315T CHaT positive cells in 4M Male mouse spinal cord samples. Therefore, our results warrant further investigation of the role of both glycolysis and HK1 in ALS motor neuron death and disease progression.

How known mutations of GABAA receptors affect their surface trafficking

Mu Lab

Gamma-aminobutyric acid type A (GABAA) receptors are the primary inhibitory ion channels of the human central nervous system mediating fast inhibitory neurotransmission and maintaining the excitation-inhibition balance in the mammalian brain. Any loss of function of this receptor is a prominent cause of genetic epilepsies. As of today, the many anti-epileptic drugs already existing mostly focus on the suppression of the symptoms and have many side effects. Furthermore, about one-third of epilepsy patients are resistant to current drug treatment. There is thus an urgent need to better understand the molecular mechanisms of epilepsies and offer new therapeutic strategies. This proposal seeks to elucidate the molecular mechanisms underlying pathogenic GABAA receptor loss of function and to explore new therapeutic strategies to correct their function. Our team and others have shown that reduced trafficking of mutant GABAA receptors at the cell membrane is a major molecular mechanism for their loss of function. Thus, in a first aim we intend to study how known mutations of GABAA receptors affect their surface trafficking. Then in a second aim we propose to identify new molecules able to correct the malfunction of mutant GABAA receptors to enhance their surface trafficking as a novel strategy to treat genetic epilepsies. Using the brain’s own tools via small molecules constitutes a new therapeutic strategy that provides safer results and is more likely to avoid off-target effects.

Investigating the roles of phosphorylation and splice variation in the Drp1 variable domain

Ramachandran Lab

Dynamin-related protein 1 (Drp1) is a mechanoenzyme responsible for mitochondrial division. Drp1's activity in mitochondrial membrane remodeling is regulated by an auto-inhibitory ~130- residue stretch known as variable domain (VD), which is intrinsically disordered yet facilitates the specific binding of Drp1 to cardiolipin-enriched mitochondrial membranes. Moreover, the VD is subject to alternative splicing and various post-translational modifications, including phosphorylation, whose functions are unclear. Our preliminary results reveal an understanding of the four splice variants of VD and shed light on their tendency to interact with cardiolipin enriched membranes. In vitro analysis of the VD in isolation using SEC-MALS and circular dichroism reaffirms the monomeric, disordered conformation of the VD for all splice variants, as well as for phosphomimetic and phosphodeficient mutants of the shortest variant. Probing VD dynamics through fluorescence quenching of the single Trp in this region gives insights into the interactions with cardiolipin-enriched liposomes as VD gains helicity.

The deficiency of branched chain ketoacid dehydrogenase kinase (BCKDK), the negative regulator of BCAA catabolism, induces various mitochondrial defects and exacerbates aSynuclein pathology

Qi Lab

Parkinson’s Disease (PD) is a neurodegenerative disease characterized by progressive motor deficits that arise as a result of dopaminergic neuron degeneration. Neuronal degeneration is accompanied by the aggregation of aSynuclein, which in turn disrupts various critical cell processes, including mitochondrial function. Mitochondrial dysfunction is widely implicated as a contributing factor in neuronal cell death owing to its importance in cell energetics, though there is still much to be discovered concerning its role in PD pathogenesis. GWAS have indicated several mitochondrial genes as potential risk factors in Parkinson’s Disease, and among these, we have identified the branched chain amino acid (BCAA) metabolic pathway as a promising mechanism in the development of PD (Nalls et al., 2019). Our studies confirm the dysregulation of the BCAA metabolic pathway in mouse models and patient samples of PD. Additionally, we find that deficiency of branched chain ketoacid dehydrogenase kinase (BCKDK), the negative regulator of BCAA catabolism, induces various mitochondrial defects and exacerbates aSynuclein pathology. We also identify glycolysis as an affected pathway that may influence the course of disease.

Proteolytic cleavage, for all intents and purposes, deactivates PR-B’s anti-inflammatory action, allowing for uninhibited pro-inflammatory signaling and pro-labor mechanisms

Gonzalez-Vicente Lab

Progesterone (P4) is the most important hormone for the maintenance of pregnancy, and its withdrawal, whether functional or systemic, initiates parturition. Targeting factors that lead to inflammatory events within Progesterone Receptor (PR) pathways may help keep a fetus at risk for preterm birth (PTB) in the uterus longer, increasing chances of survival and decreasing potential negative consequences. However, gaps in our understanding of these highly complex progesterone signaling pathways remain. Canonically, once liganded, the full length PR (PR-B), is known to have anti-inflammatory actions by interfering with AP-1’s transcriptional activity. This is a major pro-gestational drive during the quiescent period of pregnancy. However, the anti-inflammatory activity associated with PR-B decreases drastically as pregnancy nears its culmination—the mechanism of which is currently unknown. We postulate that when an “Inflammatory Threshold” is met during pregnancy, PR-B’s activity may decline due to proteolytic cleavage. This cleavage likely occurs near the 164^th residue of PR-B, directly distal to the B-Upstream Segment (BUS) that is unique to PR-B. We hypothesize that this proteolytic cleavage, for all intents and purposes, deactivates PR-B’s anti-inflammatory action, allowing for uninhibited pro-inflammatory signaling and pro-labor mechanisms. This research addresses a major physiological knowledge-gap, i.e.: the mechanism by which pregnancy is both maintained through quiescence and concluded through parturition. The goal of this study is to determine the mechanism of PR-B cleavage and its association with the onset of labor. This will allow us to understand the function of the progesterone receptors in the human myometrium, and provide specific, targetable mechanisms for potential preterm birth therapies. Ultimately, this research will contribute to the development of effective PR-based therapies to prevent PTB. These aims will be achieved using several innovative model systems including genetically modified human myometrial hTERT cell lines, frozen/fixed and explant cultures of gravid human myometrium, mouse models of inflammation-induced PTB and physiological pregnancy phenotypes, and optimized assays using validated reagents to measure 1) relative abundance of specific mRNAs; 2) abundance of specific proteins; 3) physical interaction pathways between PRs and AP-1 subunits; 4) specific sub-cellular localizations of PR-B; and 5) development of model systems in human myometrial cell lines and transgenic mice, within which PRs are mutated to observe functionality.

Solving the high resolution structure of the NBCe1-b/IRBIT complex by Cryo electron microscopy (Cryo-EM)

Vahedi-Faridi Lab

NBCe1-b, an electrogenic sodium bicarbonate Na+/HCO₃-cotransporter, is one of several transmembrane proteins that interacts with the IRBIT protein (IP3R-binding protein released with inositol 1,4,5-triphosphate). IRBIT interactions with NBCe1-B have been proposed to alter NBCe1-B activity in the pancreas, brain, and other organs. Thus, elucidating these interactions at the atomic level would be of immense value in understanding the physiological outcome.Our goal is to solve the high resolution structure of the NBCe1-b/IRBIT complex by Cryo electron microscopy (Cryo-EM). Elucidation of these structures, both individually and complexed, will further our understanding the mechanism of the Na/HCO₃-transporter molecule, its conformational interactions and activation by the IRBIT ligand. We transfected recombinant FLAG-tagged NBCe1-B into HEK cells. By surface biotinylation we show that the recombinant protein inserts into the plasma membrane. We have also expressed recombinant Strepll-tagged IRBIT in HEK cells which has been co-transfected together with NBCe1-B . Additionally we expressed and purified milligram amounts of recombinant histidine-tagged IRBIT in E.coli. This purified protein will used to create a stable binding complex with recombinant NBCe1-B as well as with the above-mentioned synthetic NBCe1-B peptide.

Analyzing how the forces involved in TDP-43 LLPS were affected by the phosphomimetic substitutions, and supplemented our findings with additional experiments.

Surewicz Lab

Amyloid inclusions made up of TAR DNA-binding protein of 43 kDa (TDP-43) appear in many neurodegenerative conditions, including amyotrophic lateral sclerosis, frontotemporal lobar degeneration, and Alzheimer’s disease. The protein within these inclusions is phosphorylated, primarily at the C-terminal residues S403, S404, S409 and S410. There is increasing evidence that the ability of TDP-43 to undergo liquid-liquid phase separation (LLPS) and form membraneless compartments (“droplets”) is linked to amyloid aggregation and contributes to pathogenesis. The effect of TDP-43 C terminal phosphorylation on its LLPS, however, has not been well-studied. To explore this question, we introduced phosphomimetic substitutions (Ser->Asp) at pathologically relevant sites in TDP-43’s low complexity domain, a part of the protein known to drive its phase separation, to generate a doubly-substituted protein (2xphos) and a quadruply-substituted protein (4xphos) and investigated how the LLPS of the protein changed. We then used coarse-grained simulations to analyze how the forces involved in TDP-43 LLPS were affected by the phosphomimetic substitutions, and supplemented our findings with additional experiments.

Vahedi-Faridi Lab

NH₃/NH₄+transport is essential to nitrogen metabolism throughout all domains of life. Bacterial ammonia transporters (Amt proteins, AmtB, Rh50) have paralogs in yeast (MEP proteins) and humans (Rhesus proteins RhAG, RhBG, RhCG). The Boron lab showed that the Rh channel proteins AmtB (bacterial), RhAG, RhBG, and RhCG (human) are permeable for both CO2 and NH3. Previous structural work suggested that NH3 moves through the three monomeric pores of the trimeric channel protein. To address the issue of how Rh proteins conduct non-polar gases, we planned to trap noble gases (Xe, Kr) in the channels of a high quality AmtB crystal. Crystallization trials were set up based on already published conditions. AmtB crystals were pressurized with Xe gas at 100 - 300 psi in a Hampton Xenon chamber and frozen in liquid nitrogen afterwards for diffraction studies. Crystals diffracted to 1.8 Å at the Synchrotron at the APS, Argonne, IL. Several complete anomalous data sets (at 1.45 Å wavelength) of Xe derivatized AmtB crystals were collected. Two data sets exhibited significant anomalous signal resulting in the localization of 13 Xe atoms with varying occupancies. These data in combination with Molecular Dynamics simulations provide the first evidence for any physiological function of the central pore of AQPs or Rh-proteins, and represent a significant advance in channel biology (manuscript in preparation).

Sexually dimorphic expression of the angiotensinogen gene(Agt)in the S3 subsegment of rat proximal tubules

The renin-angiotensin systems (RAS), systemic and intrarenal, play a critical role in blood pressure regulation. Sexual dimorphism affects various organ systems including the RAS, with males being more sensitive to the hypertensive actions of angiotensin II (AngII). We hypothesize that rat kidney single-cell transcriptomes could inform sex differences in the intrarenal RAS. We integrated publicly available datasets to create a single-cell RNA-seq map of the rat kidney including 3 females and 3 males to study sexually dimorphic gene expression in different epithelial cells of the nephron. We identified sex-differentially expressed genes (DEGs) in proximal tubules (PT), thick ascending limbs (TAL), distal convoluted tubules (DCT), collecting ducts (CD), and thin limbs (Thin. Limbs). The number of upregulated genes in females and males respectively were as follows: PT-F) 86, TAL-F) 7, DCT-F) 3, CD-F) 4, Thin.limbs-F) 4, PT-M) 104, TAL-M) 29, DCT-M) 26, CD-M) 36, Thin.limbs-M) 36. From these genes, a KEGG pathway enrichment analysis was conducted which revealed that females exhibited increase expression of genes involved in glutathione biosynthesis and metabolism. In contrast, males displayed higher expression of genes associated with mineral transport and the Na,K ATPase γ-subunit. As PTs concentrate the larger number of DEG, we analyzed the PT subpopulations. Here, the number of upregulated genes in females and males respectively were: PT.S1-F) 39, PT.S2-F) 37, PT.S3-F) 73, PT.S1-M) 67, PT.S2-M) 57, PT.S3-M) 105. In PT.S3, females maintained elevated glutathione biosynthesis and metabolism, while males exhibited sustained mineral transport and elevated expression of the Na,K-ATPase γ-subunit. Males also showed increased expression of angiotensinogen and proteases involved in angiotensin peptide metabolism. Increased expression of elevated glutathione biosynthesis and metabolism in females suggests increased protection against oxidative stress, while increased expression of mineral transport activity and demand suggests higher energy demand. Males also have increased expression of Agt and proteases capable of metabolizing angiotensin peptides, which suggests male susceptibility to hypertensive actions of AngII.

Elucidation of the Native Structure of Microtubule-Bound Drp1 Using Cryo Electron Tomograph

Ramachandran Lab

Dynamins are a class of large, mechanochemical GTPases, and Drp1 (dynamin-related protein 1) is a pivotal member of this superfamily. Drp1 is primarily known for its crucial role in mitochondrial fission, a process that ensures the dynamic and balanced distribution of mitochondria within eukaryotic cells. Drp1 exerts its fission-inducing function by constricting and dividing mitochondria through a process highly dependent on its interaction with microtubules. Microtubules also play a role in the intracellular transport and positioning of mitochondria for division. They play a crucial role in orchestrating the recruitment and localization of Drp1 to mitochondria during fission events. To better comprehend the intricate relationship between Drp1 and microtubules, it is essential to elucidate the native structure of Drp1 when bound to microtubules. Cryo-electron tomography (Cryo-ET) emerges as a powerful tool in this context, offering the ability to visualize biological structures in their native state at high resolution. This study aims to shed light on the structural intricacies of the Drp1-microtubule interaction, providing insights into the mechanisms that govern mitochondrial fission and various other cellular processes. Herein, we used a mutant of Drp1 (named 4EA) engineered to tightly bind to microtubules. The mouse embryonic fibroblast (MEF) cell line was used for its expression and binding with microtubules. Expression and microtubule binding of the 4EA mutant were confirmed by visualizing both fixed and live cells by confocal microscopy. After confirming the binding, the cells were processed for cryo-ET experimentation using a customized pipeline.

UNDERSTANDING RTK FAMILY DIFFERENECES IN EPHA1 & EPHA2 HOMO DIMERIZATION BY MOLECULAR DYNAMICS SIMULATION

Buck Lab

Ephrin Receptors are receptor tyrosine kinases which play a critical role in cellular growth, differentiation and cell motility. Overexpression of particularly the EphA2 receptor has been reported in several different cancers and it is known that a cancer stimulating and supportive function can be activated in a ligand independent manner which it different from its normally cell retraction/cell movement restrictive function as a tyrosine kinase. In Eph receptors, ephrin ligand binding shifts the monomer-dimer equilibrium towards larger-scale receptor clustering through a conformational change in the extracellular region (ECR), but also tyrosine kinase activity is associated with dimerization of the transmembrane (TM) domain. EphA2, known for its ligand independent signalling, and the lesser cancer associated, EphA1 differ significantly in their primary sequence in their transmembrane (TM), and in their membrane proximal domains: two extracellular Fibronectin III domains (FN1&2) and the juxtamembrane (JM) region. In order to understand the role of these domains in EphA1 and –A2 and their differences, we carried coarse grained molecular dynamics (CG-MD) simulations of their association in/at an 80% POPC, 15% PS and 5% PIP2 containing membrane. The recently published Martini 3 potential function was used in order to increase sampling. The JM domain has several basic residues (K/R) that are closely positioned to the membrane surface, while the surface of the EphA2 2nd FN domain has also been reported to interact with the lipid bilayer and these interactions are confirmed here. Moreover, we find that the FN domains and the JM region also function in synergy with the TM domain. Overall, our simulations add emphasis to the emerging importance of TM and TM proximal domains of Eph receptors and provide testable models for the signal transduction mechanisms and different activated states.

Steltzer Lab

Systemic and Cellular Metabolic Remodeling in a Mouse model of HCM

Hypertrophic cardiomyopathy (HCM) is the most prevalent inherited cardiomyopathy, with majority of cases caused by pathogenic variants of MYBPC3. Patients present with reduced ejection fraction (EF) due to contractile dysfunction caused by a hypertrophied left ventricle (LV). There is emerging evidence that abnormal contractile function is closely related to metabolic remodeling in cardiomyocytes. However, this theorized metabolic rewiring has not been evaluated at a systemic nor cellular level in animal models of HCM. Characterization of an HCM mouse model (MYBPC3-/- global knockout) showed reduced fat stores, elevated blood ketone levels, and that whole-body respiration was more reliant on carbohydrate catabolism compared to wild-type (WT) counterparts. Upon a two-hit metabolic challenge to exploit glycolytic capacity with a treadmill and substrate utilization restriction using a high-fat diet (HFD), MYBPC3-/- mice on HFD were more reliant on fatty acid catabolism at the whole-body level, and had a higher glycolytic capacity than WT mice on a HFD. Further, mitochondria isolated from the LV of MYBPC3-/- mice showed lower basal and β-oxidation linked state 3 respiration compared to WT mitochondria. Altogether, these data suggest that MYBPC3-/- hearts have an increased reliance on glucose catabolism due to reduced LV mitochondrial function. The metabolic rewiring at the mitochondrial level of MYBPC3-/- mice affects systemic metabolism, evidenced by whole-body characterization data.

Mutant Huntingtin mimetic protein-like polymer blocks mitochondrial damage and slows onset of neuropathology in vivo

Abstract: Recently, a neuroprotective peptide (HV3-TAT) which blocks the binding of valosin containing protein (VCP) to mutant Huntingtin protein (mtHtt) has been shown to prevent neuronal mitochondrial autophagy (mitophagy) in the R6/2 mouse model of Huntington’s disease (HD). However, peptides alone are limited by poor pharmacokinetic profiles due to lack of stability, vulnerability to proteolysis, and increased clearance. To overcome these challenges, a proteomimetic platform for scaffolding peptides has been developed, termed the Protein-Like Polymer (PLP). PLPs are globular, peptide brush polymer structures, synthesized here from norbornenyl-HV3 monomers via graft through ring-opening metathesis polymerization (ROMP). The resulting neuroprotective PLPs were shown to maintain bioactivity in cell-based in vitro assays by successfully inhibiting a mitochondrial pathway. In this manner, PLP and HV3-TAT peptide both rescue HD mouse striatal cells. However, PLP is significantly resilient to in vitro enzyme, serum and liver microsome stability assays which render the peptide ineffective. Further, when compared head-to-head in vivo, PLPs demonstrated an over 2000-fold increase in circulation detection compared to the peptide alone, with PLP exhibiting an elimination half-life of over 150 hrs. In addition, the PLP is biocompatible and is well tolerated (1.69 mg/kg/day, 8 weeks) as evidenced by blood compatibility, organ pathology and blood toxicity analysis in normal mice. In vivo efficacy studies in HD transgenic mice (R6/2) confirmed the superior bioactivity of PLPs compared to free peptide through both behavioral and neuropathological analyses. These data support the conclusion that PLP prevents pathologic VCP/mtHtt binding in HD animal models, exhibits enhanced efficacy over the parent, free peptide and implicates the PLP as a platform with potential for translational CNS therapeutics.

Exploring ligand modulation of the human versus mouse 5-HT3A receptor

Chakrapani Lab

The 5-hydroxytryptamine type 3 receptor (5-HT₃R), the only ionotropic receptor in the serotonin receptor family, has important roles in gut modulation and gut-brain communication. These receptors are made up of five subunits, which together form an extracellular domain (ECD), transmembrane domain (TMD), and intracellular domain (ICD). 5-HT₃ receptors can be found as homomers composed of five A subunits, or heteromers composed of the A subunit and four other possible subunits: B, C, D, or E. While this receptor has links to diseases such as irritable bowel syndrome (IBS), obsessive compulsive disorder (OCD), and schizophrenia, current treatments for these diseases target the more ubiquitously expressed A subunit of this pentameric channel. Antagonists of this receptor, known as setrons, are currently on the market to provide relief for patients experiencing IBS or nausea while undergoing chemotherapy. Unfortunately, secondary effects such as constipation and ischemic colitis are a risk while using these antagonists due to their causing full inhibition of receptor activity. Partial agonists of 5-HT_3AR are thus becoming a promising alternative. However, current research has shown differences in response to some partial agonists between the human 5-HT_3AR and mouse 5-HT_3AR. The importance of mice in drug development and the progression of clinical research is unavoidable. Understanding the structural components of these receptors which lead to these different responses to partial agonists is therefore critical for future drug design and clinical studies.

Oxygen-offloading rate from mouse Red Blood Cells of genetically diverse strains

Boron Lab

O2 diffusion across red blood cell (RBC) membranes is a critically important physiological process for the maintenance of life. Our previous work on murine RBCs (bioRxiv. doi:10.1101/2020.08.28.265066) showed that (a) the genetic deletion of aquaporin-1 (AQP1) and the Rh complex (mainly RhAG) together reduce O2 permeability of RBC membranes (P_M,O2) by ~55%, and (b) the double knockout (dKO) of AQP1 and RhAG plus pCMBS (sulfhydryl reagent and nonspecific inhibitor of membrane proteins that is nonetheless excluded from the RBC interior) reduces P_M,O2 by ~91%. In order to identify the unknown membrane protein(s) that may function as O₂ channels and contribute to the missing 91% – 55% = ~36% of P_M,O2, we use comparative physiology. Using stopped-flow absorbance spectroscopy to monitor the hemoglobin (Hb) absorbance spectrum, we studied the oxygen-offloading rate from hemoglobin (kHbO2) of RBCs from genetically diverse mouse strains. We compared two different mouse strains purchased from Jackson Labs—C57BL/6J and BALB/cJ with our standard mouse strain that ultimately derives from mice received from UCSF—C57BL/6Case. We found that, compared with C57BL/6Case, C57BL/6J mice have a k_HbO2 for intact RBCs that is ~22% higher (N=18, 9 male plus 9 female, p<0.01), and a kHbO2for pure hemolysate that is ~7% lower (N=12, 6 male plus 6 female, p<0.01). Comparing with C57BL/6Case, BALB/cJ mice have a k_HbO2 for intact RBCs that is ~9% lower (N=18, 9 male plus 9 female, p<0.01), and a k_HbO2of pure hemolysate that is ~10% higher (N=12, 6 male plus 6 female, p<0.01). Because changes in k_HbO2 of pure hemolysate are opposite in direction to changes in kHbO2of intact RBCs, differences in Hb cannot contribute to the observed differences in O₂-offloading rates in the two Jackson strains. Likewise, changes of mean corpuscular volume (MCV) and mean corpuscular hemoglobin concentration (MCHC), together, would only produce minor percent changes of k_HbO2 in either C57BL/6J or BALB/cJ mice. Thus, it is likely that the differences in k_HbO2 for intact RBCs among the three mouse strains reflect major differences in the O₂ permeabilities of the RBC membranes. Planned comparative analyses of morphology (e.g., major diameter of biconcave discs), proteomics, lipidomics, and genomics (for deduced amino-acid sequences of proteins) will provide valuable insight into the contribution of specific membrane proteins and lipids to the relatively large differences in k_HbO2 of intact RBCs between C57BL/6J and BALB/cJ mice.

Tajima Lab

Abstract: Kainate receptors (KARs) are a class of ionotropic glutamate receptors (iGluRs) and are abundantly expressed in the brain. They regulate both excitatory and inhibitory transmission. While the overall architecture of KARs shows structural similarities to other iGluRs like AMPA and NMDA receptors, the desensitized KARs demonstrate strikingly different conformation, indicating the gating mechanism of KARs is unique. To date, how the activation of KARs is distinct from other iGluRs is not well understood. Here, combining single particle cryo-electron microscopy (cryo-EM), microscopic electrophysiological recordings and cysteine biochemistry, we identify conformational changes at the ligand binding domain interface between the two constitute dimers in GluK2 KARs upon activation. We will further analyze the dynamics by fluorescence resonance energy transfer assay to better understand the KAR gating.